Adducts of mitomycin C with nucleotides.

说明(申请人提供)：丝裂霉素C(MC)是一种用于癌症化疗临床的天然抗肿瘤抗生素。MC在肿瘤细胞中产生六种不同的DNA加合物。其中一种是DNA链间交联物，这是MC对互补的DNA链进行双官能团烷基化的结果；另一些是药物的加合物，只与一条DNA链相连(单加合物和链内交联物)。本提案的广泛、长期的目标是阐明六种加合物中的每一种与MC的细胞毒性/抗肿瘤活性的关系。此外，将使用Fanconi的贫血(FA)细胞来研究MC氧化还原循环产生的活性氧物种(ROS)的假定毒性作用。该提案的第一个目的是确定已知的FA细胞对MC的超敏主要是由于ROS的产生还是由于药物形成DNA加合物。这些研究将更好地确定法尼奥尼贫血细胞独特敏感性的生化基础，并提供对丝裂霉素活性的见解。其他特定目的用于检测和表征MC的不同DNA加合物所特有的生化反应。在此背景下，我们将研究在细胞培养中活的EMT6肿瘤细胞中加合物的不同修复速度。在亚细胞系统中，通过构建针对不同MC加合物的定点修饰的91聚体寡核苷酸双链，并将其用作uvrABC外切核酸酶的底物或哺乳动物全细胞提取物中存在的修复酶的底物，将在亚细胞系统中研究交联物和单加合物的不同核苷酸切除修复模式。同样的底物将被用来在细胞提取物中寻找“加合物结合蛋白”，这种蛋白是针对交联链的。也将使用合成的加合物-模板-底物作为底物，研究不同的丝裂霉素单加合物对DNA聚合酶对病变旁路的不同抑制作用。后者的实验旨在确定单加合物5和6细胞毒性异常低的基础。这项研究将使人们更好地了解MC的抗肿瘤活性和毒理学的分子和生物化学基础，以及更广泛地说，其他DNA交联剂，目前用于癌症化疗的一种主要药物。

DESCRIPTION (provided by applicant): Mitomycin C (MC) is a natural antitumor antibiotic used in the clinic for cancer chemotherapy. MC generates six different DNA adducts in tumor cells. One of these is a DNA interstrand cross-link, as a result of bifunctional alkylation of the complementary DNA strands by MC, while the others are adducts of the drug, linked to only one strand of DNA (monoadducts and an intrastrand crosslink). The broad, long-range objective of the present proposal is to elucidate the relationship of each of the six adducts to the cytotoxicity/antitumor activity of MC. In addition, the putative toxic role of reactive oxygen species (ROS), generated by redox cycling of MC, will be examined using Fanconi's Anemia (FA) cells. The first aim of the proposal is to determine whether the known hypersensitivity of FA cells to MC is primarily due to ROS generation or to DNA adduct formation by the drug. These studies will better define the biochemical basis of the unique sensitivities of Faneoni's anemia cells, as well as providing insights into the activities of the mitomycins. The other specific aims serve to detect and characterize biochemical responses unique to the different DNA adducts of MC. In this context we shall investigate differential rates of repair of the adducts in living EMT6 tumor cells in cell culture. Differential modes of nucleotide excision repair of cross-link and monoadducts will be investigated in subcellular systems by constructing 91-mer oligonucleotide duplexes modified site-specifically with the various MC adducts, and using these as substrates for uvrABC excinuclease, or for the repair enzymes present in a mammalian whole cell extract. The same substrates will be used to search for "adduct binding proteins", specific for the cross-link, in cell extracts. Differential inhibition of lesion bypass by DNA polymerases by the different mitomycin monoadducts will also be investigated, using synthetic adduct-template-primer complexes as substrates. These latter experiments are aimed at identifying the basis for the unusually low cytotoxicity of the monoadducts 5 and 6. This research will lead to a better understanding of the molecular and biochemical basis of the antitumor activity and toxicology of MC and, more generally, of other DNA cross-linking agents, a major class of drugs currently used in cancer chemotherapy.